Human Apolipoprotein A-I is the major protein component of (HDL), which is an important lipoprotein in blood. It is synthesized by the liver and intestine and is responsible for the physiological function of HDL in the blood; the removal of cholesterol from peripheral tissues, carrying it back either to the liver or to other lipoproteins, by a mechanism known as “reverse cholesterol transport” (RCT).
The clear correlation between elevated levels of serum cholesterol and the development of coronary heart disease has been repeatedly confirmed, based on epidemiological and longitudinal studies.
Hence, Apo A-I in HDL is thought to have an anti-inflammatory function and to restrain the occurrence and development of CHD. Furthermore, Apo A-I has shown to decrease the Low Density Lipoproteins (LDL) level in the blood and is known to bind to endotoxins, thus having a major role in the anti-endotoxin function of HDL.
The “protective” role of HDL and Apo A-I as its major component has been confirmed in a number of studies making Apo A-I a promising candidate (particularly as part of a reconstituted HDL) for applications in atherosclerosis treatment, acute coronary syndrome treatment (ACS), anti-inflammation treatment, antitoxin treatment, liver-targeting drugs, etc.
Human blood plasma is nowadays collected in large amounts and processed to individual fractions some of which contain apolipoprotein A-I. The fractions may be produced by ethanol fractionation according to a procedure originally developed in the United States and known as Cohn or Cohn-Oncley methods [E. J. Cohn et al., J. Am. Chem. Soc. 68, 459-475, 1946; J. L. Oncley et al., J. Am. Chem. Soc. 71, 541-550, 1949]. Plasma fractions containing apolipoproteins may also be produced by a variant of this method, the Kistler-Nitschmann procedure [H. Nitschmann et al., Helv. Chim. Acta 37, 866-873, 1954; P. Kistler and H. Nitschmann, Vox Sang 7, 414-424, 1962]. Both methods are based on differential precipitation in alcohol.
Various approaches have been described in the literature to recover apolipoprotein A-I from ethanol precipitation fractions:
U.S. Pat. No. 5,089,602 for instance, relates to the preparation of apolipoproteins from fractions of human blood plasma or serum by resuspending the fractions in an aqueous buffer solution in the pH range 3 to 5 or 6 to 9. Undesirable contaminants are precipitated by addition of a short chain aliphatic alcohol. Use is made of buffers containing high ethanol concentrations (68-96% ethanol) for precipitating contaminants. Potential aggregation of lipoproteins is inhibited through elevated temperature, slightly alkaline pH, or by the addition of chaotropic agents or surface-active substances, which is subsequently removed by gel filtration. An anion-exchange chromatography step is included to bind the contaminants, while the apolipoproteins pass through.
Kim et al., Manufacturing and Shelf Stability of Reconstituted High-density Lipoprotein for Infusion Therapy, Biotechnology and Bioprocess Engineering 16, 785-792 (2011) disclose the use of extraction buffers containing urea in a concentration of 6 M.
A combination of the two above mentioned methods is described in WO 98/07751 for recovering apolipoprotein A (Apo A-I) or apolipoprotein E (ApoE) from human plasma. Said document discloses a process comprising at least one pre-purification step through the use of an extraction buffer containing 8 M urea and a further purification step using anion-exchange chromatography. In WO 98/07751, 1% recovery of Apo A-I resulted from experiments using an extraction buffer comprising 20% ethanol. WO2009/025754 teaches methods to separate Apo A-I from alpha-1 antitrypsin including the use of lower ethanol concentrations (8 to 14%) to precipitate Apo A-I.
Other methods such as ultra-speed centrifuge and high performance liquid chromatography (HPLC) are also used to recover Apo A-I. However, the preparation of Apo A-I is extremely time-consuming and only minor quantities of Apo A-I can be prepared by these methods. These methods are therefore not suitable for an industrial production of Apo A-I.
Thus, there remains a need for a method for extracting and recovering Apo A-I from Apo A-I containing protein fractions, which is suitable for large-scale production and which allows for acquiring Apo A-I in a high yield.